12alpha-methyl pregnene derivatives



United States Patent ice 3,169,978 lzd'METHYL PREGNENE DERIVATIVESPatrick A. Diassi,'Westfield, Pacifico A. Principe, South River, andJosef Fried, Princeton, N.J., assignors to Olin Mathieson ChemicalCorporation, New York,

N.Y., a corporation of Virginia No'Drawing. Original application May 31,1963, Ser. No. 284,313. Divided and this application Mar. 5, 1964, Ser.No.'354,495

3 Claims. (Cl. 260-397.3)

This application is a'div-ision of application Serial No. 284,313, filedMay 31, 1963.

This invention relates to a new process for the production of steroidsand to new intermediates produced *dn the process.

Steroids of the formula wherein R and R represent an llfi-hydroxy orll-keto structure and the 1,2-position is saturated or double bonded, aswell as the 16 and/or 21 esters thereof, are physiologically activesubstances which possess glucocorticoid, antiinflamm-atory andprogestational activity and which are also useful in the production ofthe 16,17-acetal and ketal derivatives disclosed in US. Patent No.3,048,581. This invention rel-ates to a new process for producing suchcompounds.

According to the process of this invention, the starting materials arel2a-lower alkyl-l lfl-hydroxyp-rogesterone, 12a-loweralkyl-llB-ketoprogesterone or the corresponding l-dehydroprogesterones.These starting materials are subjected to the enzymes of Streptomycesroseochromogarms in an aqueous medium containing a source of nitrogenousfactors and an assimilable source of carbon and energy, in the presenceof oxygen, and the l6a-hydroxy steroid of the formula lower alkyl thusformed is recovered.

3,159,978 Patented reb. I6, 1965 The compound of Formula II is thensubjected to the action of an aluminum alkoxide to form a newintermediate of the formula (III) CH3 l lower alkyl (i=0 The preferredconditions include an aluminum lower alkoxide, especiallyaluminurn tbutoxide, an elevated temperature somewhat in excess of room temperatureup to about reflux temperature, an organic solvent, especially anaromatic hydrocarbon of the benzene series such as toluene or xylene,and an inert atmosphere especially nitrogen.

Next, the compound of Formula III is subjected to the action of theenzymes of the microorganism Wojnowicia graminis, Ophiobolusherpotrichus or Aspergz'llus niger in an aqueous medium containing asource of carbon and energy, in the'presence of oxygen, and is therebyconverted to a new compound of the formula I V) onion lower alkyl +=Opreferably in an organic hydrocarbon solvent of the benzene series inthe presence of an organic base such as pyridine at about roomtemperature to obtain a compound of the formula (V) CHgO-IOWGI alkanoyllower alkyl =0 R -OH R I a rated or double bonded. 12ot-methyl compoundsare preferred throughout.

The following examples are illustrative of the invention with alltemperatures being expressed on the centigrade scale. A method for thepreparation of the starting materials is included in the examples. Ineach instance, a 12cclower alkyl compound other than the 12a-methylcompound illustrated may be utilized if a lithium alkyl other thanlithium methyl, e.g., lithium ethyl, is used in the preparation of the12a-alkylprogresterone used as starting material.

EXAMPLE I (a) Preparation of 9a-flu0ro-11ketoprogesterone3,20-bis-ethylene ketal A mixture of 10 g. of9a-fluoro-1l-ketoprogesterone, 350 ml. of benzene, 80 ml. of ethyleneglycol and 200 mg. of paratoluenesulfonic acid monohydrate is refluxedwith stirring for 72 hours. The reaction mixture is then cooled to roomtemperature and neutralized with sodium bicarbonate solution. The phasesare separated and the aqueous layer reextracted with additional amountsof benzene. The combined benzene extracts are washed with water, driedover sodium sulfate and evaporated to dryness in vacuo. The cruderesidue on crystallization from acetone-hexane yields about 11 g. of theessentially pure bis-ethylene ketal melting at about 179-182".Recrystallization of this material from methanol gives an analyticalsample of the following properties: M.P. about 189-190; [th, 25.

Analysis.Calcd. for C H O F (434.53): C, 69.10; H, 8.12. Found: C,69.19; H, 8.18.

(b) Preparation of JZa-methyI-J 1 ketoprogesterone 3,20-bis-ethyleneketal A solution of 9u-fluoro-ll-ketoprogesterone 3,20-bisethylene ketalg.) in benzene (100 ml.) is treated with an ethereal solution of lithiummethyl (150 ml., 13.5 mg. of lithium metal/ml). The solution is stirredfor 4 hours at room temperature and then the excess lithium methyl isdecomposed by the addition of ice. Chloroform (300 ml.) is added, andthe mixture is washed several times with water, dried over sodiumsulfate and evaporated in vacuo. Trituration of the residue with hexanegives about 4.2 g. of l2a-methyl-ll-ketoprogesterone 3,20-bis-ethyleneketal, M.P. about 135-138". A second crop of crystals (about 2.4 g.,M.P. about 124-130) is obtained on concentrating the hexane motherliquor. Crystallization from methanol gives an analytical sample meltingat about 139-142", [1x1 8.8 (c. 0.716 in CHCl Analysis.-Calcd. for C H O(430.56): C, 72.50; H, 8.90. Found: C, 72.71; H, 8.90.

(c) Preparation of 12ot-methyl-lJfl-hydroxyprogesterone3,20-bis-ethylene ketal A solution of l g. of1Za-methyl-llketoprogesterone 3,20-bis-ethylene ketal in 50 ml. of drytetrahydrofuran is heated under reflux with l g. of lithium aluminumhydride for 18 hours. Ice is added to the cooled solution to decomposeexcess reagent and then a saturated aqueous solution of sodium sulfateis added with stirring until the precipitated aluminum salts are formedin a slurry. The clear ether solution is decanted off and the organicmaterial is washed twice with chloroform. The combined organic extractsare dried over sodium sulfate and then evaporated in vacuo. The residueis dissolved in 10 ml. benzene and absorbed on a column of 30 g. ofalumina. Elution with benzene (900 ml.) and chloroform-benzene (1:9, 500ml), followed by crystallization from acetonehexane, yieldsIZa-methyl-llfi-hydroxyprogesterone 3,20- bis-ethylene ketal (about 660mg.) melting at about 169- 175. Crystallization from acetone-hexaneafi'ords an 4 analytical sample which melts at about 177-l79; [otl ll.5(c. 1.24in CHCl will Analysis.Calcd. for C H O (432.58): C, 72.19; H,9.32. Found: C, 72.30; H, 9.20.

(d) Preparation of 1Za-methyl-ll{i-hydroxyprogesterone Analysis.Calcd.for C H O (344.48): C, 76.70; H,

9.32. Found: C, 76.59; H, 9.41.

(6) Preparation of 1 Za-methyI-I 1/3, 16u-dihydr0xyprogesterone AStreptomyces roseochromogenus (Waksman No. 3689, The Institute ofMicrobiology, Rutgers University, New Brunswick, New Jersey) culture ismaintained on Gould agar (agar, 20 g.; glucose, 10 g.; yeastextract,"2.5 g.; K HPO 1 g.; distilled water to 1 liter). Inoculum forthe first flask stage is prepared by suspending the surface growth from'each of 2 two week old agar slant culture with 5 ml. of an 0.01% Duponol(Merck Index, 7th ed., 1960) solution. One milliliter portions ofinoculum are used to inoculate ten 250 ml. Erlermeyer flasks, eachcontaining 50 ml. of the following medium (A):

' Grams Soybean meal 20 Glucose 30 Soybean oil 2.2 Calcium carbonate 2.5

Distilled water to 1 liter.

I of 45 250 ml. Erlenmeyer flasks, each containing ml.

of the same medium A. At the time of inoculation of these flasks, 25 mg.of steroid is added to each flask using 0.25 ml. per flask of a mg./ml.solution of 12arnethyl-llfi-hydroxyprogesterone inN,N-dimethylformamide. A total of 1.125 g. of steroid is therebyfermented. After inoculation and supplementation the flasks are thenincubated under the same conditions as described above. At approximatelyhours after steroid addition, the culture broths are harvested. Thecontents from each flask are pooled and the pooled broth is thenadjusted to pH 4.5 using 12 N H 50 The acidified broth is then filteredthrough a Buchner-Seitz clarifying pad apparatus. The filtrate (3000ml.) is collected and extracted three times with one liter portions ofchloroform. The combined chloroform extracts are washed twice with 1.5l. of water and then evaporated to dryness in vacuo. Crystallization ofthe residue from acetone-hexane gives about 825 mg. of 12a-methyl-l1p,16ot-dihydroxyprogesterone having a melting point about 208209, [111+164 (chloroform) A313, 242 mu (e=15,800) Analysis.-Calcd. for C H O(360.48): C, 73.30; H, 8.95. Found: C, 73.32; H, 8.91.

(7'') Preparation of 12ot-methyl-A -pregnadiene-IIB-ol- 3,20-di0ne Asuspension of 640 mg. of l2a-methyl-l1/3,16c:-dihydroxyprogesterone and1.5 g. of aluminum tert-butoxide 5 in 180 ml. of dry toluene is refluxedunder nitrogen for two hours. The mixture is then cooled and washedsuccessively with 2 N hydrochloric acid, water, 5% sodium bicarbonateand water and then evaporated to dryness in vacuo. The residue iscrystallized from acetone-hexane to yield 475 mg. of l2a-methyl-A-pregnadiene-llfl-ol- 3,20-dione having 'a melting point about 224-226,[a] +258 (chloroform) M1,} 244 mu (e-26,000)

Analysis-Calcdlfor C I-1 (342.46): C, 77.15; H, 8.83. Found: C, 77.11;H, 8.73.

(g) Preparation of IZa-methyI-J6-dehydr0cortic0 ster0ne 21-acetate AWojnowicia graminis culture (Centraal Bureau voor Schimmel Cultures,Baarn, The Netherlands) is main tained on Gould agar (agar, 20 g.;glucose, 10 g.; yeast extract, 2.5 g.; K HPO 1 g.; distilled water to 1liter). Inoculum for the first flask stage is prepared by suspending thesurface growth from each of 2 two weekold gear slant cultures with 5 ml.of an 0.01% Duponol (Merck Index, 7th ed., 1960) solution. Onemilliliter portions of inoculum are used to inoculate ten 250 ml.Erlenmeyer flasks, each containing 50 ml. of medium (A): Soybean meal,20 g.; glucose, 30 g.; soybean oil, 2.2 g.; calcium carbonate, 2.5 g.;distilled water to 1 liter. The flasks are. then incubated at 25 C. on arotary shaker (280 cycles per minute, 2 inch radius) for 96 hours. After96 hours, a 10% transfer (by volume) is made to each of 50 250 ml.Erlenmeyer flasks, each containing 50 ml. of the following sterilizednutrient medium (B): dextrose, 10 g.; cornsteep liquor, 6 g.; NH.,H PO 3g.; Difco yeast extract, 2.5 g.; CaCO 2.5 g.; and distilled water to 1liter. At the time of inoculation of these flasks, 5 mg. of steroid isadded to each flask using 0.25 ml. per flask of a 20 mg./ml. solution of1Za-methyl-A -pregnadiene- 11B-ol-3,20-dione in N,N-dimethyl formamide.A total of 250 mgs. of steroid is thereby fermented. After inoculationand supplemention of the flasks with steroid, the flasks are thenincubated under the same conditions as described above.

After 96 hours of further incubation, the contents of the flasks arepooled and filtered through a Seitz clarifying pad. The flasks, myceliumand pad are washed with successive 50 ml. portions of warm water. Thecombined filtrate and washings have a volume of 2800 ml. This isextracted with three 3.3 1. portions of chloroform which are combined,washed twice with 5.0 1. portions of water and evaporated to dryness invacuo. The residue is dissolved in a mixture of 12 ml. of dry pyridineand 4 ml. of acetic anhydride and left at room temperature for fourhours. Upon addition of ice water, crystals of12amethyl-l6-dehydrocorticosterone 21 acetate separate which arefiltered, Washed with water and dried. This compound has a melting pointabout 190192 C., [0;] +197 (chloroform) A313; 242 my (c=22,000)

Analysis.Calcd. for C H O (400.50): C, 71.97; H, 8.05. Found: C, 71.77;H, 8.14.

By substituting either a culture of Ophiobolus herpetrichus (CentraalBureau voor Schimmel Cultures, Baarn, The Netherlands) or a culture ofAspergillas niger (ATCC 9142) for the culture of Wojnowicia graminis inthe above procedure and otherwise following the same technique,12a-methyl-16-dehydrocorticosterone is similarly obtained.

(h) Preparation of 12a-methyl-16a-hya'r0xyhydr0c0rti-- sone 21 -acetateTo a stirred solution of 25.4 mg. of 12ot-methyl-16dehydrocorticosterone ZI-aceta'te in 4 ml. of benzene containing 0.2 ml.of pyridine a solution of 16.7 mg. (0.066

mmol.) of osmium tetroxide in 0.82 ml. of benzene is added dropwise overa ten minute period. The solution is stirred at room temperature andafter 45 minutes a precipitate separates. The stirring is continued for2% hours and then 4 ml. of an aqueous solution containing .36 g. each ofsodium sulfite and potassium bicarbonate which is free of oxygen isadded followed by 2 ml. of methanol. The mixture 'is then stirred undernitrogen for 3 hours, filtered and washed thoroughly with chloroform andwarm tetrahydrofuran. The filtrate is washed with water and evaporatedto dryness in vacuo. Crystallization of the residue from acetone-hexanegives 10 mg.

of 12a-methyl-1 6a-hydroxydrocortisone 21-acetate having a melting pointabout 194-196" C.

(i Preparation of12u-methyl-16a-hydroxyhydracortis0ne ,To a stirredsolution of 50 mg. of 12a-methyl-16a-hydroxyhydrocortisone 21-acetate in10 ml. of methanol (oxygen free) is addedl ml. of 10% K 00 (oxygen free)and the'mixture stirred at room temperature under nitrogen' for twohours. This solution is then neutralized with 1 m1. of 10% acetic acidand diluted with water whereupon crystals separate. They are filtered,washed with water and dried to give 35 mg. of12e-methyl-16ahydroxyhydrocor'tisone.

EXAMPLE 2 V (a) Preparation of IZa-meIhyZ-Il-ketoprogesterone A solutionof 300 mg. of 12a-methyl-1l-ketoprogesterone 3,20-bis-ethylene ketal in10 ml. methanol and 1 ml. of 8% sulfuric acid is heated under reflux for4'hours. The mixture is then diluted with water, the precipitated solid(about 210 mg., M.P. about 154156) collected and crystallized fromacetone-hexane. The resulting sample of 12a-methyl-1l-ketoprogesteronemelts at about 155157; [oz] +227 (c. 1.36 in CHC1 8&3? 236 mp (15,800);W 11i 5.88, 5.96, 6.18;].

Analysis.--Calcd. for C H 0 (342.46): C, 77.15; H, 8.83. Found: C,76.60; H, 8.76.

Similarly, by substituting12a-ethyl-1l-ketoprogesterone-3,20-bis-ethylene ketal for the IZa-methylsteroid in the above procedure, 12a-ethyl-1l-ketoprogesterone isobtained. Furthermore, any other bis ketal derivatives can be similarlyhydrolyzed to yield the same free 3,11,20- triketone derivatives.

(b) By substituting 12a-methyl-1l-ketoprogesterone for12a-methyl-1lp-hydroxyprogesterone in Example l(e) and following thesame procedure described in parts (e) through (i) of that example, thereare obtained successively 12u-methyl-16a-hydroxy-1l-ketoprogesterone,120L- methyl-A -pregnadiene-3,11,20-trione, 12a-methyl-Apregnadiene-21-ol-3,11,20-trione, IZa-methyI-A-pregnadiene-21-ol-3,l1,20-trione 21-acetate,12a-methyl-16a-hydroxycortisone ZI-acetate and12amethyl-16a-hydroxycortisone.

EXAMPLE 3 (a) Preparation of 1Za-methyl-A -pregnadiene-3J1,20- trione Toa solution of mg. of 12a-methyl-11-ketoprogesterone in 10 ml. of dioxaneis added 60 g. of 2,3-dichloro- 5,6-dicyanobenzoquinone and the mixturerefluxed under nitrogen for 6 hours. After cooling, the precipitated2,3- dichloro5,6-dicyanohydroquinone is filtered and washed withdioxane. The filtrate is diluted with an equal volume of chloroform andpoured onto a column of 10 g. of Woelm neutral alumina. Elution withchloroform followed by evaporation of the solvent and crystallization ofthe residue gives 12u-methyl-A fl-pregnadiene-3,11,20= trione.

(b) Preparation of 12a-methyl-16a-hydroxyprednisone By substituting theproduct of part (a) above in Example 1(a) and following the sameprocedure described in parts (e) through (i) of that example there areobtained successively 12a-methyl-A -pregnadiene-16a-ol-3 ,11,20- trione,1Za-rnethyl-A -pregnatriene-3,11,20-trione, 12amethyl-A r -pregnatrienc3,11,20 trione, IZa-methyl- A i -pregnatr'iene-21-ol-3,l1,20-trioneZI-acetate, 12arnethyl-16u-l1ydroxyprednis0ne ZI-acetate and Hot-methyl-16a-l1ydroxyprednisone.

EXAMPLE 4 Preparation of 12a-methyl-A -pregnadiene- I1fl-0l-3,20-di0neBy following the procedure of Example 3 in its entirety,

but substituting IZa-methyl-llfi-hydroxyprogesterone as' What is claimedis:

1. 12u-methyl-A -pregnadiene-11fi,16a-diol-3,20-dione. 2. 1Zoc-methyI-A-pregnatriene-3,11,20-trione.

3. 1Za-methyl-Ahh -pregnatriene-1 lfi-ol-3,20-dione.

References Cited by the Examiner UNITED STATES PATENTS 3,014,905 12/61Yang et a1 260-239.55 3,048,581 8/62 Fried 260-23955 3,056,730 10/62Laskin 19551 3,080,388 3/63 Thomas et a1. 260317.3

OTHER REFERENCES Nagarajan et al.: Can.'J. Chem. vol. 39 (1961), pages1274-1278.

LEWIS GOTTS, Primary Examiner.

2. 12A-METHYL-$1,4,16-PREGNATRIENE-3,11,20-TRIONE.